The hemagglutinin protein (HA) gene of avian influenza virus was amplified by polymerase chain reaction (PCR) from the recombinant plasmid pVAX1-HA, and subcloned into eukaryotic expression vector pmcDNA3.1+. The HA gene was identified by sequencing. The recombinant plasmids were transformed into attenuated Salmonella typhimurium SL7207, and the recombinants were designated as SL7207 (pmcDNA3.1-HA). In vitro and in vivo experiments showed that the plasmid stability of pmcDNA3.1-HA is apparently higher than pcDNA3.1-HA in SL7207. In order to compare the immune response induced by those two recombinant bacteria, BALB/c mice were immunized orally with them at the dosage of 2 x 10(9) CFU respectively. Both SL7207 (pcDNA3.1-HA) and SL7207 (pmcDNA3.1-HA) initiated HA-specific mucosal antibodies in immunized mice. Furthermore, commercial ISA brown chickens were immunized with SL7207 (pcDNA3.1-HA) and SL7207 (pmcDNA3.1-HA) at the dosage of 5 x 10(9) CFU and boosted two weeks later with the same dose. Intestinal mucosal immune responses were observed and their levels were significantly higher than that of negative and positive control. The result of protective immunity showed that the chicken immunized with SL7207 (pmcDNA3.1-HA) had the protective rate of 79.3%, higher than that of SL7207 (pcDNA3.1-HA) with 56.7%. In summary, the DNA vaccine delivered by attenuated Salmonella typhimurium has good immunogenicity. A novel mucosal DNA vaccine was developed and could be useful for controlling the infection and epidemic of avian influenza in the poultry.